Characterization of plasmids in a sucrose-fermenting strain of Escherichia coli.

A multiply drug-resistant strain of Escherichia coli isolated from a patient in Bangladesh was shown to carry four types of plasmids based on size differences. One type carries a gene or genes for sucrose fermentation.     

DNA-DNA homology among lactose- and sucrose-fermenting transconjugants from Lactococcus lactis strains exhibiting reduced bacteriophage sensitivity.

DNA-DNA homology between a reduced bacteriophage sensitivity (Rbs+) probe and DNA from both Rbs+ and Rbs- Lactococcus lactis strains was examined. Homology was detected between the probe and five plasmids (pCI750, pCC34, pEB56, pNP2, and pJS88) isolated from lactose-positive Rbs+ transconjugants and between the probe and genomic DNA of a sucrose-positive Rbs+ transconjugant. Additionally, hybridizations conducted between the probe and plasmids reported to encode abortive bacteriophage infection indicated homology with pTR2030 but not with pBF61 and pGBK17. The results suggest that a common genetic determinant(s) may be present in a variety of lactococcal plasmids coding for Rbs+.     

Characterization of thermodynamic diversity between transmissible spongiform encephalopathy agent strains and its theoretical implications.

Some transmissible spongiform encephalopathy (TSE) (or "prion") strains, notably those derived from bovine spongiform encephalopathy, are highly resistant to total inactivation by heat. When three TSE strains derived from sheep with scrapie were heated, little inactivation took place at low temperatures, but at higher temperatures, considerable inactivation occurred. The temperature at which substantial inactivation first occurred varied according to TSE strain, and it was calculated to be 70 degrees C for the 22C strain, 84 degrees C for ME7, and 97 degrees C for 22A by fitting the data to a model based on competition between a destructive and a protective reaction. However, PrP(Sc) from mice infected with a range of TSE strains retained similar resistance to proteinase K digestion after heating to below or above these temperatures, showing that the properties of PrP(Sc) responsible for proteinase resistance do not correlate with those conferring thermostability on the TSE agent. The simplest explanation of these data is that the causal agent contains a macromolecular component that is structurally independent of the host, that it varies covalently between TSE strains, and that it is protected by other macromolecular components. The model is in accord with the virino hypothesis, which proposes a host-independent informational molecule protected by the host protein PrP.     

DNA-DNA homology among lactose- and sucrose-fermenting transconjugants from Lactococcus lactis strains exhibiting reduced bacteriophage sensitivity

DNA-DNA homology between a reduced bacteriophage sensitivity (Rbs+) probe and DNA from both Rbs+ and Rbs- Lactococcus lactis strains was examined. Homology was detected between the probe and five plasmids (pCI750, pCC34, pEB56, pNP2, and pJS88) isolated from lactose-positive Rbs+ transconjugants and between the probe and genomic DNA of a sucrose-positive Rbs+ transconjugant. Additionally, hybridizations conducted between the probe and plasmids reported to encode abortive bacteriophage infection indicated homology with pTR2030 but not with pBF61 and pGBK17. The results suggest that a common genetic determinant(s) may be present in a variety of lactococcal plasmids coding for Rbs+.     

Characterization of MR (P) strains of Drosophila melanogaster: the number of intact P elements and their genetic effect.

To study the effect of mutagenic/carcinogenic agents on P-element transposition, the P strains used should be defined, especially with respect to the number of intact and functional P elements present. In this investigation, the relation between the number of complete P elements present in dysgenic males and P-insertion mutagenesis was studied in several MR (P) strains. The main conclusions from this investigation are: (1) Complete P elements can be present in the genome without genetic activity (even in a 'dysgenic' cross). As a consequence, the number of complete P elements present in particular dysgenic flies, is not necessarily an indication of their dysgenic genetic activity. (2) The MR-h12/Cy strain carries two complete P elements, one on the X chromosome without and one on the MR chromosome with genetic activity (making this strain most suitable for studies on P-transposition mechanisms).     

Similarity of rpoB gene sequences of sucrose-fermenting and non-fermenting Corynebacterium diphtheriae strains

During the last decades, the majority of Brazilian Corynebacterium diphtheriae isolates were shown to be capable to metabolize sucrose, sometimes leading to erroneous identification as a non-diphtheric Corynebacterium species. The sequencing of the polymorphic region of the RNA polymerase beta subunit-encoding gene (rpoB) is an important taxonomic tool for identification of corynebacteria. The present study aimed to investigate the rpoB gene polymorphic features of sucrose-fermenting and non sucrose-fermenting strains. The results showed that sucrose-fermenting strains presented rpoB gene polymorphic regions with more than 98% similarity with the sequences deposited in the gene bank corresponding to non sucrose-fermenting strains. Data indicate that sucrose-fermenting isolates may act as a variant of C. diphtheriae biotype mitis. In addition we alert that sucrose-fermenting strains should not be discarded as contaminants mainly in countries where the possibility of isolation of this variant is higher.     

Characterization and translation of transmissible gastroenteritis virus mRNAs.

Three protein species were identified in purified transmissible gastroenteritis virus particles (strain Purdue). They are thought to represent constituents of the peplomer (E2; molecular weights of 280,000 and 240,000), the envelope (E1; molecular weights of 28,000, 31,500, and 33,000), and the nucleocapsid (N; molecular weight of 48,000). In infected cells, proteins with molecular weights of 195,000 (E2), 48,000 (N), and 28,000 (E1) were detected. Tunicamycin, an inhibitor of N glycosylation, prevented the appearance of polypeptides with molecular weights of 195,000 and 28,000 in infected cells; instead, proteins with molecular weights of 160,000 and 25,000 were observed. One minor and five major mRNA species were detected in porcine cells after infection. Their size was determined to be 23.6 kilobases (kb) (RNA1), 8.4 kb (RNA3), 3.8 kb (RNA4), 3.0 kb (RNA5), 2.6 kb (RNA6), and 1.9 kb (RNA7). The RNAs were translated in vitro. RNA7 was shown to code for the N protein. Although complete separation of RNA6 could not be achieved, it was shown to encode an unglycosylated (molecular weight of 25,000) precursor of E1 (molecular weight of 28,000). RNA4 was translated into a nonstructural protein with a molecular weight of 24,000. Translation of RNA3 resulted in proteins with molecular weights of 250,000 and 130,000 and smaller molecules which could be precipitated with a monoclonal antibody directed against E2.     

Characterization of the progeny of X-ray irradiated males from two Drosophila virilis strains differing in genetic instability.

Significant effects of X-ray treatment on the increase in the number of phenotypic variations, two visible mutations, and chromosome aberrations were found in the progeny of irradiated males from the D. virilis laboratory stock that is capable of hybrid dysgenesis syndrome induction. This effect is much more pronounced than in the progeny of irradiated males from strong wild-type strains studied. A correlation between genetic instability and chromosome radiosensitivity was outlined. The mechanism of this phenomenon and the possibilities of using the property of genome instability for the productive induction of gene and chromosome damage in radiation mutagenesis experiments are discussed.     

Germ line transmission of autonomous genetic elements in transgenic mouse strains

Upon microinjection into fertilized mouse eggs of circular molecules of plasmid pPyLT1 carrying the gene encoding the large T protein of polyoma virus within bacterial vector sequences, autonomous circular plasmids were stably maintained in low copy numbers in transgenic strains. These plasmids could be rescued in E. coli by transfection. Integrated forms could be detected neither in somatic tissues, nor in spermatozoa. Efficiency of paternal or maternal transmission was close to 100%. The plasmids had lost or had extensively rearranged the polyoma sequences. In addition, they had acquired defined segments of genomic mouse DNA, which might be responsible for correct segregation of daughter copies at both mitosis and meiosis (centromeric function).     

Biofilm-producing abilities of Salmonella strains isolated from Turkey

In the present study the biofilm-forming characteristics of 99 serotyped (DMC strains) and 41 genus level-identified (IS strains) Salmonella strains originating from Turkey were investigated. The strains were selected based on their ability to show the biofilm morphotype on Congo red agar plates. In addition, all strains were evaluated with regard to properties related to forming pellicle structures, physical differences of pellicles, any changes in the media associated with the formation of pellicles, and the presence of cellulose within the formed biofilm matrix as determined using 366 nm UV light. The Salmonella Typhimurium DMC4 strain was the best producer of biofilm grown on polystyrene microtiter plates (optical density at 595 nm: 3.418). In subsequent experiments industrial process conditions were used to investigate different morphotyped Salmonella strains’ biofilm-forming capability on stainless steel, a commonly preferred surface for the food industries, and on polystyrene surfaces. The effect of other important industrial conditions, such as temperature (5, 20, 37°C), pH (4.5, 5.5, 6.5, 7.4) and NaCl concentration (0.5, 1.5, 5.5, 10.5%) on the production of biofilm of the different morphotyped Salmonella strains (DMC4; red, dry and rough morphotyped S. Typhimurium, DMC12; brown, dry and rough morphotyped S. Infantis, DMC13; pink, dry and rough morphotyped S. subsp. Roughform) were also assessed. On the other hand, pH values exhibited variable effects on biofilm-forming features for different Salmonella strains on both polystyrene and stainless steel surfaces.     

Characterization of antibiotic resistance genes linked to class 1 and 2 integrons in strains of Salmonella spp. isolated from swine

The aim of this study was to characterize the antibiotic resistance profiles, the integron-associated resistance determinants, and the potential ability of transferring these determinants by conjugation in Salmonella enterica isolated from swine. Fifty-four strains of Salmonella spp. were isolated from healthy swine. The percentages of resistance, determined by the plate dilution method were as follows: oxytetracycline (41%), streptomycin (39%), sulphamethoxazol+trimethoprim (19%), enrofloxacin-ciprofloxacin (13%), and amoxicillin (0%). The most important resistance serovars were Salmonella Branderburg, Salmonella Derby, Salmonella Typhimurium, and Salmonella Heidelberg. The oxytetracycline-resistant strains amplified the genes tetA (36%), tetB (64%); and the strains resistant to streptomycin and trimethoprim amplified the genes aadA1 (100%) and dfrA1 (100%), respectively. None of the fluoroquinolone-resistant strains amplified the gene qnr. Ten strains amplified the class 1 integron harboring the cassette aadA1. Six strains amplified the class 2 integron harboring the cassettes dfrA1, sat1, and aadA1. The conjugation assays showed that 2 strains transferred the tetA and aadA1 genes and the class 1 integron to a recipient strain. Taken together, the results obtained in this study show a high percentage of resistance in and the presence of integrons in strains of S. enterica isolated from swine. This information should support the implementation of regulations for the prudent use of antimicrobial agents in food-producing animals.     

Transfectability of rough strains of Salmonella typhimurium.

Cells of rough (but not smooth) strains of Salmonella typhimurium become competent for transfection by phage P22 deoxyribonucleic acid after treatment with 0.1 M CaCl2. The yield of infectious centers is about 10(-8) per genome equivalent of deoxyribonucleic acid. However, different sorts of rough strains vary in their ability to become competent in a fashion that can be correlated with the level of the genetic block in cell wall lipopolysaccharide synthesis. The most amenable strains are blocked by defects in the addition of galactose units I and II of the lipopolysaccharide by the inability to synthesize uridine 5'-diphosphate-galactose (galE point mutants and gal deletion mutants). Strains blocked only in the addition of galactose I, glucose I, or heptose II have low levels of transfectability, whereas strains with either more complete or more deficient lipopolysaccharide core are not competent for transfection. When normal lipopolysaccharide synthesis is restored either genetically or by furnishing exogenous galactose (galE point mutants that can still use it), the cells are not longer competent for transfection.     

R plasmids with thermosensitive transferability in Salmonella strains isolated from humans.

Temperature dependence of transfer was examined with ten R plasmids originating from clinical isolates of Salmonella. Six of the plasmids were thermosensitive upon transfer, five of which were originally harbored in S. typhimurium and the remaining one in S. derby. One of these plasmids, pNR502, which conferred resistance to kanamycin, streptomycin (Sm) and tetracycline (Tc) on its host was stably maintained both in Salmonella and Escherichia coli at either 30, 37, or 43 C. Another plasmid, pNR516, which was resistant to chloramphenicol, sulfathiazole, Sm and Tc, was slightly unstable only at 43 C. The remaining four plasmids, pNR503, pNR510, pNR512 and pNR514, conferred resistance to Sm and Tc. Of these plasmids, the former two were stably maintained at both 30 and 37 C, but were unstable at 43 C. The latter two were slightly unstable at the lower temperatures and considerably unstable at 43 C. Kinetics of the transfer of the plasmid pNR503 revealed that the efficiency of transfer of the plasmid between E. coli strains was affected not only by the temperature of the conjugation but also by the preincubation temperature of the donor culture before the conjugation.     

Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent.

Transmissible mink encephalopathy (TME) has been transmitted to Syrian golden hamsters, and two strains of the causative agent, HYPER (HY) and DROWSY (DY), have been identified that have different biological properties. During scrapie, a TME-like disease, an endogenous cellular protein, the prion protein (PrPC), is modified (to PrPSc) and accumulates in the brain. PrPSc is partially resistant to proteases and is claimed to be an essential component of the infectious agent. Purification and analysis of PrP from hamsters infected with the HY and DY TME agent strains revealed differences in properties of PrPTME sedimentation in N-lauroylsarcosine, sensitivity to digestion with proteinase K, and migration in polyacrylamide gels. PrPC and HY PrPTME can be distinguished on the basis of their relative solubilities in detergent and protease sensitivities. PrPTME from DY-infected brain tissue shared solubility characteristics of PrP from both uninfected and HY-infected tissue. Limited protease digestion of PrPTME revealed strain-specific migration patterns upon polyacrylamide gel electrophoresis. Prolonged proteinase K treatment or N-linked deglycosylation of PrPTME did not eliminate such differences but demonstrated the PrPTME from DY-infected brain was more sensitive to protease digestion than HY PrPTME. Antigenic mapping of PrPTME with antibodies raised against synthetic peptides revealed strain-specific differences in immunoreactivity in a region of the amino-terminal end of PrPTME containing amino acid residues 89 to 103. These findings indicate that PrPTME from the two agent strains, although originating from the same host, differ in composition, conformation, or both. We conclude that PrPTME from the HY and DY strains undergo different posttranslational modifications that could explain differences in the biochemical properties of PrPTME from the two sources. Whether these strain-specific posttranslational events are directly responsible for the distinct biological properties of the HY and DY agent strains remains to be determined.     

Characterization of lysine decarboxylase-negative strains of Salmonella enterica serovar Enteritidis disseminated in Japan

Salmonella enterica serovar Enteritidis is one of the leading causes of food-borne diseases in Japan. Typically, Salmonella spp. test positive for lysine-decarboxylase. However, the number of isolates of serovar Enteritidis without lysine-decarboxylase activity increased in Japan in 2003. Among 109 strains from distinct outbreaks, 10 lacked lysine-decarboxylase activity. Nine of the ten lysine-decarboxylase-negative strains showed quite similar pulsed-field gel electrophoresis profiles. Their lysine-decarboxylase phenotype was recovered by introduction of the cadBA locus from an lysine-decarboxylase-positive strain. Although the cad loci of the lysine-decarboxylase-negative strains seemed to be intact without any insertion sequences, cadC, a positive regulator of cadBA, had a single-base deletion at the same position, the 973rd base (cytosine), in all the nine lysine-decarboxylase-negative strains, whereas the wild-type cadC gene has a 1542 bp coding region (514 amino acids). This deletion was expected to produce a truncated (338 amino acids) form of CadC due to a frameshift. Because CadC senses environmental cues such as external pH and lysine through its putative C-terminal periplasmic domain, it is likely that the truncated CadC is not sensitive enough to external signaling to activate the cadBA operon, resulting in loss of the lysine-decarboxylase activity. Our results suggest that dissemination of these genetically closely related strains of serovar Enteritidis accounts for the unusual increase in the isolation of lysine-decarboxylase-negative strains.     

Comparison of properties between virulent and attenuated strains of transmissible gastroenteritis virus.

Strains of transmissible gastroenteritis (TGE) virus possessing different pathogenicity were examined for stability to digestive enzymes and acid, and growth at various temperatures. In growth experiments, virus titer obtained at 37 degrees C were about equal between attenuated and virulent strains, but titers attained by the attenuated strain were higher at 30 degrees C. The attenuated virus multiplied at 28 degrees C, but the virulent virus did not at this temperature. The virulent virus was significantly stable to trypsin and pepsin, but the attenuated virus was inactivated rapidly by these proteolytic enzymes. No significant differences were observed in stability to acid between the attenuated and virulent strains. At different pH, both lost their infectivity more rapidly at 37 degrees C than at 22 degrees C.     

Characterization of Salmonella and possible interfering strains using GC profiling and factor analysis

Fatty acid methyl ester data of Salmonella and potentially interfering strains obtained by derivatization gas chromatography were investigated with principal components and factor-discriminant analysis. Salmonella strains could be distinguished using both data-handling techniques. The biochemical basis for this characterization was found to be in strong agreement with the higher mass range part of pyrolysis — direct chemical ionization mass spectrometric data representing diglyceride information. The quality of the analytical procedure as judged by longer term reproducibility and component assignment was found to be good. In combination with sophisticated data-handling methods this derivatization gas chromatographic approach can be a useful diagnostic tool for Salmonella characterization.     

Plasmids and transposable elements in Salmonella wien.

The plasmids from six clinical strains of Salmonella wien have been characterized. All the S. wien strains were found to carry three types of plasmids: an IncFI R-Tc Cm Km Ap (resistance to tetracycline, chloramphenicol, kanamycin, and ampicillin) plasmid, either conjugative or nonconjugative, of large size (90 to 100 megadaltons); an R-Ap Su Sm (resistance to ampicillin, sulfonamide, and streptomycin) plasmid of 9 megadaltons; and a very small (1.4 megadaltons) cryptic plasmid. The characteristics of conjugative R plasmids, recombinant between F'lac pro and the FI nonconjugative plasmid, indicated that regions coding for the donor phenotype were present on this plasmid. The molecular and genetic features of the R plasmids were very close to those described for the R plasmids isolated from S. wien strains of different origin. This fact supported the hypothesis of a clonal distribution of this serotype in Algeria and Europe. The analysis used to identify transposable elements showed the presence of only TnA elements, which were located on both the R-Tc Cm Km Ap and R-Ap Su Sm plasmids. They contained the structural gene for a TEM-type beta-lactamase and had translocation properties analogous to those reported for other TnA's.